Vol 11, Issue 12, 2018 Online - 2455-3891 Print - 0974-2441 ANALYSIS REACTIVITY OF PUNICA GRANATUM POLYPHENOLS TO THE OSTEOCALCIN, BONE MORPHOGENETIC PROTEIN-2, AND COLLAGEN TYPE-1 EDRIZAL BURHAN 1,2 , BERGMAN THAHAR 3 , TRIMURNI ABIDIN 4 , DEDDI PRIMA PUTRA 5 , BASRI A GANI 6 1 Department of Orthodontic, Dentistry Faculty, Baiturahmah University, Padang, Indonesia. 2 Department of Doctoral Program, Dentistry Faculty, North Sumatera University, Medan, Indonesia. 3 Department of Orthodontic, Dentistry Faculty, Padjajaran University, Bandung, Indonesia. 4 Department of Conservative, Dentistry Faculty, North Sumatera University, Medan, Indonesia. 5 Department of Pharmacy, Andalas University, Padang, Indonesia. 6 Department of Oral Biology, Dentistry Faculty, Syiah Kuala University, Banda Aceh, Indonesia. Email: basriunoe@unsyiah.ac.id Received: 11 September 2018, Revised and Accepted: 24 October 2018 ABSTRACT Objective: Punica granatum (PG) contains anthocyanins which are useful as antioxidants, anti-inflammatory, and prevent cancer, while also increasing bone cell proliferation and osteoblasts differentiation in bone remodeling. Methods: The reactivity of osteocalcin protein markers with PG polyphenol fractionation hydrogels observed using enzyme-linked immunosorbent assay, the degree of reactivity was determined by optical density at 550 nm. Results: PG butanol fraction has better reactivity compared to the total extract, ethyl, and hexane fraction. Based on the reactivity distribution, bone morphogenetic protein (BMP)-2 and collagen Type-1 had a dominant distribution compared to osteocalcin, but the theses proteins had a strong relation (r = 0.8) with probability (P < 0.05). Conclusion: PG butanol fraction had better reactivity to osteocalcin, BMP-2, and collagen Type-1 compared with total extract, hexane, and ethyl fraction. The four PG polyphenol fractionations have dominant reactivity to BMP-2. Keywords: Punica granatum, Reactivity, Osteocalcin, Bone matrix protein-2, Collagen Type-1. INTRODUCTION Pomegranate (Punica granatum [PG]) peel contains proanthocyanidin which is a family of flavonoids [1]. This active component acts as an antioxidant, anticancer, and also anti-inflammatory by inhibiting pro- inflammatory cytokines [2]. Moreover, these active components can also inhibit a number of enzymes that play a role in cell differentiation such as cyclooxygenase, lipooxygenase, cytochrome P450, phospholipase A2, ornithine decarboxylase, carbonic anhydrase, 17 beta-hydroxysteroid dehydrogenase, and serine protease [3]. PG peel extract contains various active components that differ depending on the extractor which used. The largest phenolic levels were found in butanol fraction compared to other extractors. The ethanol extract of PG peel produces a number of polyphenols which can trigger the expression of angiogenesis cells in the process of new bone formation [4]. A number of studies have reported that PG polyphenol fractionation has a significant effect on osteoblast cell viability as well as curcumin polyphenol fractionation as an immunostimulator for osteoblast expression in bone remodeling case [5]. The pomegranate peel of the Ganesh variety that extracted with ethanol and methanol was evaluated for the phenol content which contained at those extract. Methanol extract has better immunotolerant potency against pathogens than ethanol extract, while a mixture of ethanol and methanol extract has a very good antioxidant potency with phenol content in it which can act as an antibacterial either antioxidant [6]. Siddiqui et al. reported that PG can increase bone cell proliferation and osteoblast differentiation that is characterized by the expression of the runt-related transcription factor 2 (Runx2) gene. This assumption can be used as a reference for osteoporosis medication [7]. Meanwhile, PG ethanol extract can also be used as an anti-osteoporosis drug due to its ability to induce glucocorticoid hormones in osteoporosis mice model [8]. Furthermore, Bahtiar et al. reported that the use of PG polyphenol fractionation in concentrations of 50, 100, and 200 mg/kg can significantly prevent bone loss, this is related to an increase in bone calcium, particularly by increasing osteoblast [9], likewise in ovariectomy case, PG can be a stimulus to prevent bone loss [10]. The ability of PG in bone remodeling had been used to be a reference for this study, so the aim of this study was to test the ability of PG that interacts with proteins involved in bone remodeling such as osteocalcin, bone morphogenetic protein (BMP)-2, and collagen Type-1. MATERIALS AND METHODS Material This study has passed ethical clearance from the Dentistry Faculty, North Sumatra University, Medan-Indonesia. This study used PG polyphenol fractionation as the assay material to measure the degree of reactivity from osteocalcin, BMP-2, and collagen Type-1 (Abcam, Cambridge, USA) proteins. The Enzyme-linked Immunosorbent Assay (ELISA) assay will be used to the reactivity analysis PG polyphenol with the bone marker proteins as the indicator of bone remodeling. Extraction and fractionation of PG The first stage is the extraction and fractionation of PG as a test material based on methods that had been done by Arma et al. [11]. 900 g of fresh PG peel that has been cleaned and peeled off, wind dried for 2×24 h, cut into small pieces and mashed. Subsequently macerated with 96% ethanol (1:10) 9 L for 24 h and stirred in the first 6 h. On the 2 nd day, the macerate was filtered (macerate I), the pulp continued maceration with 96% ethanol (1:5) 4.5 L for 24 h. On the 3 rd day, the macerate was filtered and merged with the macerate I. Furthermore, the solvent was evaporated so that a thick extract of 1164.4 g was obtained. Fractionation Research Article © 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2018.v11i12.29683